The progression of in situ to invasive carcinoma is a poorly understood key step of breast tumorigenesis characterized by the loss of the myepithelial cell layer and basement membrane. We hypothesize that the differentiation of bipotential mammary epithelial progenitors to myoepithelial cells is progressively inhibited by signals coming from tumor epithelial cells and stromal cells such as leukocytes leading to their eventual disappearance. We propose three specific aims to test this hypothesis. Aim 1: To define the cellular composition and molecular profiles of immune cells infiltrating normal human breast tissue. Pan-leukocyte populations will be isolated from organoids and stroma of normal breast tissue samples with different parity status to dissect their role in normal breast development using immunofluorescence, FACS, and molecular profiling studies. Aim 2: To define the cellular composition and molecular profiles of immune cells infiltrating in situ and invasive breast carcinomas. Genes and pathways that may influence the differentiation of myoepithelial cells will be identified by molecular profiling of infiltrating leukocytes in in situ and invasive breast tumors. Aim 3: To test if modulating paracrine signaling pathways between leukocytes and mammary epithelial progenitors would influence myoepithelial cell differentiation and progression to invasion. Key candidate regulators of myoepithelial cell differentiation secreted by leukocytes will be validated using three dimensional co-culture and xenograft models. The long-term objective of this application is to characterize the role of immune cells in myoepithelial cell differentiation during in situ to invasive breast carcinoma progression using in vivo and in vitro model systems, and human breast tissues. The completion of this project will not only help our understanding of the role of immune cells in breast cancer, but abnormally expressed paracrine signaling in the tumor microenvironment may provide new targets for breast cancer treatment. PUBLIC HEALTH RELEVANCE: The transition of in situ to invasive carcinoma is a poorly understood key event in breast tumor progression. Immune cells are one of the cell populations that compose the tumor microenvironment and regulate cell proliferation, angiogenesis, and metastasis. Elucidating the role of leukocytes in the transition of ductal carcinoma in situ to invasive breast cancer will improve our knowledge of the role cell interactions play in tumorigenesis and will open new venues for cancer therapy and prevention.